A brain-penetrant chimeric protein combines direct glioma targeting with myeloid cell-mediated immune reprogramming

BACKGROUND: Glioblastoma (GB) is the most aggressive primary brain tumor, characterized by poor prognosis, profound immunosuppression, and limited response to current therapies. To overcome these barriers, we developed CTX-CNF1, a brain-penetrant recombinant protein that integrates direct glioma targeting with myeloid cell-mediated immune reprogramming. METHODS: CTX-CNF1 was generated by fusing Chlorotoxin (CTX), which enables selective delivery across the blood-brain barrier, with Cytotoxic Necrotizing Factor 1 (CNF1), a bacterial protein with antitumor and immunostimulatory properties. Therapeutic efficacy was evaluated in immunocompetent orthotopic GB mouse models using survival analysis, MRI, motor assessment, and immunophenotyping. RESULTS: Systemic CTX-CNF1 administration was well tolerated and significantly prolonged survival, reduced tumor burden, and preserved motor function. A substantial proportion of treated animals achieved complete tumor regression and resisted tumor re-challenge, consistent with the induction of durable antitumor immunity. Mechanistically, CTX-CNF1 promoted a pro-inflammatory reprogramming of tumor-associated myeloid cells and enhanced CD8⁺ T-cell recruitment and cytotoxicity, thereby counteracting GB-associated immunosuppression. Therapeutic efficacy was confirmed across distinct glioma models. In combination settings, CTX-CNF1 selectively enhanced the activity of immune checkpoint blockade in a context-dependent manner. CONCLUSIONS: CTX-CNF1 represents a dual-action therapeutic strategy that combines tumor targeting with immune modulation and effective brain delivery. By coupling direct antitumor effects with remodeling of the tumor immune microenvironment, CTX-CNF1 supports the development of combinatorial approaches to overcome therapeutic resistance in GB and other immune-cold tumors.